This invention relates to methods for determining the relative amount of a particular hemoglobin derivative in blood samples. More particularly, the invention concerns the determination of hemoglobin derivatives, such as glycated hemoglobin, which requires the separate measurement of total hemoglobin as well as the measurement of the hemoglobin derivative in order to determine the proportion or percentage of the derivatized hemoglobin in the blood.
The determination of the relative amount of hemoglobin that exists in the bloodstream in a particular derivative form generated by in vivo processes is of importance in a variety of medical situations. Of particular significance is the measurement of glycated hemoglobins (sometimes referred to as glycosylated hemoglobins). The measurement of glycated hemoglobin to assess the long-term blood glucose control in diabetes mellitus is being used with increasing frequency. The most important of the glycated hemoglobins in diabetes management is the derivative commonly known as hemoglobin Alc. This hemoglobin derivative is produced in vivo by the nonenzymatic reaction of glucose with hemoglobin. Glucose becomes covalently attached in a rearranged form to the amino-terminal valine residue of the beta-chain in native hemoglobin. Normal individuals have between about 3 and 6 percent of their total hemoglobin in the Alc form, while an uncontrolled diabetic individual could have as much as 3 to 4-fold higher levels of this hemoglobin derivative in the blood stream.
A variety of methods have been developed for the purpose of determining glycated hemoglobins. The conventional methods are based on diverse techniques such as cation exchange chromatography, affinity chromatography, electrophoresis, and dye complexation (hydroxyfurfural/thiobarbituric acid). These techniques are well known to be tedious, complex, requiring special technical skills, and to suffer from imprecision and nonspecificity. Very recently, monoclonal antibodies specific for the glycated N-terminal peptide residue in hemoglobin Alc have been developed which enable the performance of immunoassays to determine this hemoglobin derivative (see U.S. Pat. No. 4,647,654).
The immunoassay determination of hemoglobin Alc using monoclonal antibodies is highly specific and the procedure is significantly less tedious and complex than the prior art methods. Nevertheless, the optimal hemoglobin Alc immunoassays require that the blood sample be subject to denaturing conditions in order that the glycated N-terminal peptide residue becomes available for antibody binding (see U.S. Pat. No. 4,658,022). Typical denaturing conditions have thus far been 3 molar guanidine-HCl with heating to 56.degree. C. for 15 minutes or more.
A further limitation of the prior art techniques, including the immunoassay approach, is the requirement that a total hemoglobin measurement be conducted with each determination in order that the glycated hemoglobin level can be expressed meaningfully as a percentage. This only adds to the total time and expense of performing the assay.